Dissociation Kinetics of Actinomycin D from Individual GpC Sites in DNA

Abstract

We have examined the kinetics of dissociation of actinomycin from GpC sites in several DNA fragments containing synthetic DNA inserts, by a variation of the footprinting technique. Complexes of the ligand with radiolabelled DNA fragments were dissociated by adding a large excess of unlabelled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the GpC sites located in different sequence environments. Actinomycin dissociates more slowly from GpC sites flanked by (AT),, than A,, . T!<. Within regions of alternating AT, TGCA represents a better binding site than AGCT, and CGCA is a better binding site than GGCA. GpC sites flanked by (AC),, . (GT), present good binding sites; in this context, dissociation from CGCG is faster than from TGCA. Several studies have shown that the dissociation of actinomycin from DNA is slow, and may be an important factor for determining its biological activity [2]. Although the dissociation from synthetic DNAs, such as poly(dG-dC), is completely described by a single exponential [IS, 161, the dissociation from mixed-sequence DNAs is a complex reaction which requires at least three exponentials for its complete description, with time constants ranging over 10-1000s [2,[15][16][17][18]. This has been explained by suggesting that the multiexponential decay represents parallel dissociation of the drug from different binding sites on the heterogeneous DNA lattice, each of which possesses its own characteristic dissociation rate constant. A similar effect has been observed for the quinoxaline group of antibiotics which bind around the sequence CpG by bifunctional intercalation [19, 201. In contrast, the derivative actinomine, which lacks the peptide portion, binds to DNA with nearly the same affinity as actinomycin, yet displays much faster dissociation kinetics and has little biological activity [2]. KeywordsAlthough the fastest dissociating components must represent the interaction of the ligand with weaker sites, it is not clear whether these correspond to non-GpC sites or to a subset of GpC sites with lower affinity. Indeed, the dissociation from poly(dG-dC) is faster than the slowest component from natural DNAs 1171, suggesting that blocks of alternating GC residues do not represent the best ligand-binding site. Various studies have shown that the binding of actinomycin to GpC is influenced by the nature of the surrounding base pairs. The strongest [23]. In a series of dissociation experiments with synthetic oligonucleotides, Chen [24-271 has shown that, in general, actinomycin dissociates faster from GC sites which are preceded by G or followed by C than from other sites such as AGCA, AGCG or CGCA. The dissociation from ATATGCATAT is unusually slow (t,,, = 3000 s), while GGCC represents one of the fastest dissociating GpC sites [25]. Other studies have shown that actinomycin binds well to several non-GpC sites [28]. In particular, the sequen...

Insights into DNA‐drug interactions in the era of omics

“…[32][33] Actinomycin D intercalates its chromophore between 5 0 -GC-3 0 DNA base pairs, [34] yet not all the 5 0 -GC-3 0 sites are equivalent targets for this compound. [35] Actinomycin D antitumor activity is related to its capacity for inhibiting RNA polymerases and decrease transcription. It is used as a tool for producing rapid and specific inhibition of RNA synthesis in studies on the metabolism of messenger RNA.…”